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Metabolic fate of fructose in human adipocytes: a targeted (13)C tracer fate association study.

Varma V, Boros LG, Nolen GT, Chang CW, Wabitsch M, Beger RD, Kaput J - Metabolomics (2014)

Bottom Line: A targeted stable isotope tracer fate association method was used to analyze metabolic fluxes and flux surrogates with exposure to escalating fructose concentration.This study demonstrated that fructose stimulates anabolic processes in adipocytes robustly, including glutamate and de novo fatty acid synthesis.Furthermore, fructose also augments the release of free palmitate from fully differentiated adipocytes.

View Article: PubMed Central - PubMed

Affiliation: Division of Systems Biology, National Center for Toxicological Research, FDA, 3900 NCTR Road, Jefferson, AR 72079 USA.

ABSTRACT

The development of obesity is becoming an international problem and the role of fructose is unclear. Studies using liver tissue and hepatocytes have contributed to the understanding of fructose metabolism. Excess fructose consumption also affects extra hepatic tissues including adipose tissue. The effects of fructose on human adipocytes are not yet fully characterized, although in vivo studies have noted increased adiposity and weight gain in response to fructose sweetened-beverages. In order to understand and predict the metabolic responses of adipocytes to fructose, this study examined differentiating and differentiated human adipocytes in culture, exposed to a range of fructose concentrations equivalent to that reported in blood after consuming fructose. A stable isotope based dynamic profiling method using [U-(13)C6]-d-fructose tracer was used to examine the metabolism and fate of fructose. A targeted stable isotope tracer fate association method was used to analyze metabolic fluxes and flux surrogates with exposure to escalating fructose concentration. This study demonstrated that fructose stimulates anabolic processes in adipocytes robustly, including glutamate and de novo fatty acid synthesis. Furthermore, fructose also augments the release of free palmitate from fully differentiated adipocytes. These results imply that in the presence of fructose, the metabolic response of adipocytes in culture is altered in a dose dependent manner, particularly favoring increased glutamate and fatty acid synthesis and release, warranting further in vivo studies.

No MeSH data available.


Related in: MedlinePlus

Schematic of the 13C labeled fructose-derived metabolites in adipocytes. Adipocytes were exposed to 0.1, 0.5, 1, 2.5, 5, 7.5 or 10 mM fructose in a medium containing a baseline amount of 5 mM glucose from the initiation of differentiation for 8 days (differentiating adipocytes) or 16 days (differentiated adipocytes). 10 % of fructose was supplied as [U-13C6]-fructose for a period of 48 h before harvest. The red arrows indicate the specific fold changes in response to 5 mM fructose treatment compared to the lowest treatment of 0.1 mM fructose (13CO2, +1.15×; extracellular [13C]-glutamate, +7.2×; PC flux, −0.32; PDH flux, +3.97×; intracellular [13C]-palmitate, +4.8×; extracellular release of [13C]-palmitate −1.67×; FAS flux, +4.33×; intracellular [13C]-oleate, +2.56×; extracellular [13C]-lactate, +3×). G1P glucose 1-phosphate, G6P glucose 6-phosphate, G6PDH glucose 6-phosphate dehydrogenase, F6P fructose 6-phosphate, FAS fatty acid synthase, GAP glyceraldehyde 3-phosphate, PYR pyruvate, OAA oxaloacetate. ↑ represents increasing fold change; ↓ decreasing fold changes and the thickness of the arrow represents intensity of the fold change
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Fig6: Schematic of the 13C labeled fructose-derived metabolites in adipocytes. Adipocytes were exposed to 0.1, 0.5, 1, 2.5, 5, 7.5 or 10 mM fructose in a medium containing a baseline amount of 5 mM glucose from the initiation of differentiation for 8 days (differentiating adipocytes) or 16 days (differentiated adipocytes). 10 % of fructose was supplied as [U-13C6]-fructose for a period of 48 h before harvest. The red arrows indicate the specific fold changes in response to 5 mM fructose treatment compared to the lowest treatment of 0.1 mM fructose (13CO2, +1.15×; extracellular [13C]-glutamate, +7.2×; PC flux, −0.32; PDH flux, +3.97×; intracellular [13C]-palmitate, +4.8×; extracellular release of [13C]-palmitate −1.67×; FAS flux, +4.33×; intracellular [13C]-oleate, +2.56×; extracellular [13C]-lactate, +3×). G1P glucose 1-phosphate, G6P glucose 6-phosphate, G6PDH glucose 6-phosphate dehydrogenase, F6P fructose 6-phosphate, FAS fatty acid synthase, GAP glyceraldehyde 3-phosphate, PYR pyruvate, OAA oxaloacetate. ↑ represents increasing fold change; ↓ decreasing fold changes and the thickness of the arrow represents intensity of the fold change

Mentions: In order to determine how the [U-13C6]-fructose tracer-derived product isotopomers changed with respect to the different concentrations of fructose, multiple linear regression analyses were performed to determine the coefficients of determination (R2) and correlation coefficients (R). Fructose exposure served as the independent variable and the 13C-labeled isotopomers were the dependent or response variables. Figure S1 represents the SGBS isobolome-wide associations in a heat map using the [U-13C6]-fructose flux surrogate markers with the corresponding fructose concentration. The flux surrogate markers are positional 13C labeled intermediary metabolic products that represent specific reactions and their rates in the isobolome (i.e., the 13C labeled metabolome). The values are the amount of the fructose-derived metabolites expressed as percentages of those levels detected in control adipocytes exposed to 0.1 mM labeled fructose on days 8 or 16, the differentiating (Fig. S1-A) or fully differentiated adipocytes (Fig. S1-B), respectively. The isotopomer association patterns for palmitate and oleate showed relatively high R2 and R in differentiating (Fig. S1-A) and differentiated adipocytes (Fig. S1-B). These results indicated that the palmitate synthesis pathway and extracellular release of palmitate (limited to differentiated adipocytes) were strongly associated with the concentration of fructose in a significant manner and corroborate the lipogenic nature of fructose. For example, in differentiated adipocytes (Fig. 6b), the R2 and R values were high and significant for [13C]-palmitate content (R2 ≥ 0.950 and R ≥ 0.975), de novo palmitate synthesis via fatty acid synthase (R2 ≥ 0.974 and R ≥ 0.987), intracellular [13C]-oleate content (R2 ≥ 0.815 and R ≥ 0.903) and for the palmitate released (media [13C]-palmitate) 13C content) (R2 ≥ 0.888 and R ≥ 0.943). However, R2 and R values were low and not significantly correlated for the total cellular palmitate (peak area) in differentiated adipocytes (R2 = 0.00 and R = 0.00) (Fig. S1-B) or inversely correlated in differentiating adipocytes (R2 = 0.015 and R = −0.121) (Fig. S1-A). These results indicated that although palmitate was robustly labeled and generated from fructose, the fructose-derived intracellular palmitate is a transient product and is converted to oleate by elongation or released from the cells. Hence, the fructose-derived palmitate did not significantly alter the overall size of palmitate pool in adipocytes. On comparing the enrichment of palmitate and oleate (Figs. 5a, b, 6a, b), the enrichment of oleate is higher than that of palmitate. Oleate accumulation can be seen by the higher basal levels of labeled oleate compared to that of labeled palmitate in the control (0.1 mM fructose treated) adipocytes. Adipocytes store oleate-enriched triglycerides by elongating unlabeled palmitate into stearate, a process, as rapid as that of de novo palmitate synthesis. The increased oleate 13C labeling related to that of palmitate also indicates that palmitate is only partially, a direct precursor of newly labeled oleate. Since oleic acid gets readily incorporated into triglycerides as efficiently as its precursor (palmitate), palmitate’s elongation with fructose-derived acetyl-CoA into the stored product (oleic acid) is expected to occur in a lipogenic substrate environment, such as that observed in this study. However, based on regression analysis, palmitate enrichment correlated more strongly with increasing fructose concentrations compared to that with oleate, in this cell type (Fig. 1a, b) [(R2 = 0.950 and R = 0.975) for palmitate enrichment in differentiated adipocytes and (R2 = 0.907 and R = 0.952) in differentiating adipocytes] and [(R2 = 0.815 and R = 903) for oleate enrichment in differentiated adipocytes and (R2 = 0.850 and R = 0.922) in differentiating adipocytes respectively]. These results suggest that that there is also an attempt by the cell to simultaneously replenish the palmitate pool in the presence of increasing fructose concentrations as palmitate formed is being used up either for conversion to stearate and subsequently oleate or for release from adipocytes.Fig. 6


Metabolic fate of fructose in human adipocytes: a targeted (13)C tracer fate association study.

Varma V, Boros LG, Nolen GT, Chang CW, Wabitsch M, Beger RD, Kaput J - Metabolomics (2014)

Schematic of the 13C labeled fructose-derived metabolites in adipocytes. Adipocytes were exposed to 0.1, 0.5, 1, 2.5, 5, 7.5 or 10 mM fructose in a medium containing a baseline amount of 5 mM glucose from the initiation of differentiation for 8 days (differentiating adipocytes) or 16 days (differentiated adipocytes). 10 % of fructose was supplied as [U-13C6]-fructose for a period of 48 h before harvest. The red arrows indicate the specific fold changes in response to 5 mM fructose treatment compared to the lowest treatment of 0.1 mM fructose (13CO2, +1.15×; extracellular [13C]-glutamate, +7.2×; PC flux, −0.32; PDH flux, +3.97×; intracellular [13C]-palmitate, +4.8×; extracellular release of [13C]-palmitate −1.67×; FAS flux, +4.33×; intracellular [13C]-oleate, +2.56×; extracellular [13C]-lactate, +3×). G1P glucose 1-phosphate, G6P glucose 6-phosphate, G6PDH glucose 6-phosphate dehydrogenase, F6P fructose 6-phosphate, FAS fatty acid synthase, GAP glyceraldehyde 3-phosphate, PYR pyruvate, OAA oxaloacetate. ↑ represents increasing fold change; ↓ decreasing fold changes and the thickness of the arrow represents intensity of the fold change
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig6: Schematic of the 13C labeled fructose-derived metabolites in adipocytes. Adipocytes were exposed to 0.1, 0.5, 1, 2.5, 5, 7.5 or 10 mM fructose in a medium containing a baseline amount of 5 mM glucose from the initiation of differentiation for 8 days (differentiating adipocytes) or 16 days (differentiated adipocytes). 10 % of fructose was supplied as [U-13C6]-fructose for a period of 48 h before harvest. The red arrows indicate the specific fold changes in response to 5 mM fructose treatment compared to the lowest treatment of 0.1 mM fructose (13CO2, +1.15×; extracellular [13C]-glutamate, +7.2×; PC flux, −0.32; PDH flux, +3.97×; intracellular [13C]-palmitate, +4.8×; extracellular release of [13C]-palmitate −1.67×; FAS flux, +4.33×; intracellular [13C]-oleate, +2.56×; extracellular [13C]-lactate, +3×). G1P glucose 1-phosphate, G6P glucose 6-phosphate, G6PDH glucose 6-phosphate dehydrogenase, F6P fructose 6-phosphate, FAS fatty acid synthase, GAP glyceraldehyde 3-phosphate, PYR pyruvate, OAA oxaloacetate. ↑ represents increasing fold change; ↓ decreasing fold changes and the thickness of the arrow represents intensity of the fold change
Mentions: In order to determine how the [U-13C6]-fructose tracer-derived product isotopomers changed with respect to the different concentrations of fructose, multiple linear regression analyses were performed to determine the coefficients of determination (R2) and correlation coefficients (R). Fructose exposure served as the independent variable and the 13C-labeled isotopomers were the dependent or response variables. Figure S1 represents the SGBS isobolome-wide associations in a heat map using the [U-13C6]-fructose flux surrogate markers with the corresponding fructose concentration. The flux surrogate markers are positional 13C labeled intermediary metabolic products that represent specific reactions and their rates in the isobolome (i.e., the 13C labeled metabolome). The values are the amount of the fructose-derived metabolites expressed as percentages of those levels detected in control adipocytes exposed to 0.1 mM labeled fructose on days 8 or 16, the differentiating (Fig. S1-A) or fully differentiated adipocytes (Fig. S1-B), respectively. The isotopomer association patterns for palmitate and oleate showed relatively high R2 and R in differentiating (Fig. S1-A) and differentiated adipocytes (Fig. S1-B). These results indicated that the palmitate synthesis pathway and extracellular release of palmitate (limited to differentiated adipocytes) were strongly associated with the concentration of fructose in a significant manner and corroborate the lipogenic nature of fructose. For example, in differentiated adipocytes (Fig. 6b), the R2 and R values were high and significant for [13C]-palmitate content (R2 ≥ 0.950 and R ≥ 0.975), de novo palmitate synthesis via fatty acid synthase (R2 ≥ 0.974 and R ≥ 0.987), intracellular [13C]-oleate content (R2 ≥ 0.815 and R ≥ 0.903) and for the palmitate released (media [13C]-palmitate) 13C content) (R2 ≥ 0.888 and R ≥ 0.943). However, R2 and R values were low and not significantly correlated for the total cellular palmitate (peak area) in differentiated adipocytes (R2 = 0.00 and R = 0.00) (Fig. S1-B) or inversely correlated in differentiating adipocytes (R2 = 0.015 and R = −0.121) (Fig. S1-A). These results indicated that although palmitate was robustly labeled and generated from fructose, the fructose-derived intracellular palmitate is a transient product and is converted to oleate by elongation or released from the cells. Hence, the fructose-derived palmitate did not significantly alter the overall size of palmitate pool in adipocytes. On comparing the enrichment of palmitate and oleate (Figs. 5a, b, 6a, b), the enrichment of oleate is higher than that of palmitate. Oleate accumulation can be seen by the higher basal levels of labeled oleate compared to that of labeled palmitate in the control (0.1 mM fructose treated) adipocytes. Adipocytes store oleate-enriched triglycerides by elongating unlabeled palmitate into stearate, a process, as rapid as that of de novo palmitate synthesis. The increased oleate 13C labeling related to that of palmitate also indicates that palmitate is only partially, a direct precursor of newly labeled oleate. Since oleic acid gets readily incorporated into triglycerides as efficiently as its precursor (palmitate), palmitate’s elongation with fructose-derived acetyl-CoA into the stored product (oleic acid) is expected to occur in a lipogenic substrate environment, such as that observed in this study. However, based on regression analysis, palmitate enrichment correlated more strongly with increasing fructose concentrations compared to that with oleate, in this cell type (Fig. 1a, b) [(R2 = 0.950 and R = 0.975) for palmitate enrichment in differentiated adipocytes and (R2 = 0.907 and R = 0.952) in differentiating adipocytes] and [(R2 = 0.815 and R = 903) for oleate enrichment in differentiated adipocytes and (R2 = 0.850 and R = 0.922) in differentiating adipocytes respectively]. These results suggest that that there is also an attempt by the cell to simultaneously replenish the palmitate pool in the presence of increasing fructose concentrations as palmitate formed is being used up either for conversion to stearate and subsequently oleate or for release from adipocytes.Fig. 6

Bottom Line: A targeted stable isotope tracer fate association method was used to analyze metabolic fluxes and flux surrogates with exposure to escalating fructose concentration.This study demonstrated that fructose stimulates anabolic processes in adipocytes robustly, including glutamate and de novo fatty acid synthesis.Furthermore, fructose also augments the release of free palmitate from fully differentiated adipocytes.

View Article: PubMed Central - PubMed

Affiliation: Division of Systems Biology, National Center for Toxicological Research, FDA, 3900 NCTR Road, Jefferson, AR 72079 USA.

ABSTRACT

The development of obesity is becoming an international problem and the role of fructose is unclear. Studies using liver tissue and hepatocytes have contributed to the understanding of fructose metabolism. Excess fructose consumption also affects extra hepatic tissues including adipose tissue. The effects of fructose on human adipocytes are not yet fully characterized, although in vivo studies have noted increased adiposity and weight gain in response to fructose sweetened-beverages. In order to understand and predict the metabolic responses of adipocytes to fructose, this study examined differentiating and differentiated human adipocytes in culture, exposed to a range of fructose concentrations equivalent to that reported in blood after consuming fructose. A stable isotope based dynamic profiling method using [U-(13)C6]-d-fructose tracer was used to examine the metabolism and fate of fructose. A targeted stable isotope tracer fate association method was used to analyze metabolic fluxes and flux surrogates with exposure to escalating fructose concentration. This study demonstrated that fructose stimulates anabolic processes in adipocytes robustly, including glutamate and de novo fatty acid synthesis. Furthermore, fructose also augments the release of free palmitate from fully differentiated adipocytes. These results imply that in the presence of fructose, the metabolic response of adipocytes in culture is altered in a dose dependent manner, particularly favoring increased glutamate and fatty acid synthesis and release, warranting further in vivo studies.

No MeSH data available.


Related in: MedlinePlus